Author Correction: A naturally occurring antiviral ribonucleotide encoded by the human genome.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Publisher Correction: A naturally occurring antiviral ribonucleotide encoded by the human genome.

Change history: In the HTML version of this Letter, Extended Data Fig. 4 incorrectly corresponded to Fig. 4 (the PDF version of the figure was correct). This has been corrected online.

A naturally occurring antiviral ribonucleotide encoded by the human genome.

Viral infections continue to represent major challenges to public health, and an enhanced mechanistic understanding of the processes that contribute to viral life cycles is necessary for the development of new therapeutic strategies 1 . Viperin, a member of the radical S-adenosyl-L-methionine (SAM) superfamily of enzymes, is an interferon-inducible protein implicated in the inhibition of replication of a broad range of RNA and DNA viruses, including dengue virus, West Nile virus, hepatitis C virus, influenza A virus, rabies virus 2 and HIV3,4. Viperin has been suggested to elicit these broad antiviral activities through interactions with a large number of functionally unrelated host and viral proteins3,4. Here we demonstrate that viperin catalyses the conversion of cytidine triphosphate (CTP) to 3'-deoxy-3',4'-didehydro-CTP (ddhCTP), a previously undescribed biologically relevant molecule, via a SAM-dependent radical mechanism. We show that mammalian cells expressing viperin and macrophages stimulated with IFNα produce substantial quantities of ddhCTP. We also establish that ddhCTP acts as a chain terminator for the RNA-dependent RNA polymerases from multiple members of the Flavivirus genus, and show that ddhCTP directly inhibits replication of Zika virus in vivo. These findings suggest a partially unifying mechanism for the broad antiviral effects of viperin that is based on the intrinsic enzymatic properties of the protein and involves the generation of a naturally occurring replication-chain terminator encoded by mammalian genomes.

Lentiviral Gene Therapy Combined with Low-Dose Busulfan in Infants with SCID-X1.

BACKGROUND: Allogeneic hematopoietic stem-cell transplantation for X-linked severe combined immunodeficiency (SCID-X1) often fails to reconstitute immunity associated with T cells, B cells, and natural killer (NK) cells when matched sibling donors are unavailable unless high-dose chemotherapy is given. In previous studies, autologous gene therapy with γ-retroviral vectors failed to reconstitute B-cell and NK-cell immunity and was complicated by vector-related leukemia. METHODS: We performed a dual-center, phase 1-2 safety and efficacy study of a lentiviral vector to transfer IL2RG complementary DNA to bone marrow stem cells after low-exposure, targeted busulfan conditioning in eight infants with newly diagnosed SCID-X1. RESULTS: Eight infants with SCID-X1 were followed for a median of 16.4 months. Bone marrow harvest, busulfan conditioning, and cell infusion had no unexpected side effects. In seven infants, the numbers of CD3+, CD4+, and naive CD4+ T cells and NK cells normalized by 3 to 4 months after infusion and were accompanied by vector marking in T cells, B cells, NK cells, myeloid cells, and bone marrow progenitors. The eighth infant had an insufficient T-cell count initially, but T cells developed in this infant after a boost of gene-corrected cells without busulfan conditioning. Previous infections cleared in all infants, and all continued to grow normally. IgM levels normalized in seven of the eight infants, of whom four discontinued intravenous immune globulin supplementation; three of these four infants had a response to vaccines. Vector insertion-site analysis was performed in seven infants and showed polyclonal patterns without clonal dominance in all seven. CONCLUSIONS: Lentiviral vector gene therapy combined with low-exposure, targeted busulfan conditioning in infants with newly diagnosed SCID-X1 had low-grade acute toxic effects and resulted in multilineage engraftment of transduced cells, reconstitution of functional T cells and B cells, and normalization of NK-cell counts during a median follow-up of 16 months. (Funded by the American Lebanese Syrian Associated Charities and others; LVXSCID-ND ClinicalTrials.gov number, NCT01512888.).

Structure of the agonist-bound neurotensin receptor.

Neurotensin (NTS) is a 13-amino-acid peptide that functions as both a neurotransmitter and a hormone through the activation of the neurotensin receptor NTSR1, a G-protein-coupled receptor (GPCR). In the brain, NTS modulates the activity of dopaminergic systems, opioid-independent analgesia, and the inhibition of food intake; in the gut, NTS regulates a range of digestive processes. Here we present the structure at 2.8 Å resolution of Rattus norvegicus NTSR1 in an active-like state, bound to NTS(8-13), the carboxy-terminal portion of NTS responsible for agonist-induced activation of the receptor. The peptide agonist binds to NTSR1 in an extended conformation nearly perpendicular to the membrane plane, with the C terminus oriented towards the receptor core. Our findings provide, to our knowledge, the first insight into the binding mode of a peptide agonist to a GPCR and may support the development of non-peptide ligands that could be useful in the treatment of neurological disorders, cancer and obesity.

Panoramic view of a superfamily of phosphatases through substrate profiling.

Large-scale activity profiling of enzyme superfamilies provides information about cellular functions as well as the intrinsic binding capabilities of conserved folds. Herein, the functional space of the ubiquitous haloalkanoate dehalogenase superfamily (HADSF) was revealed by screening a customized substrate library against >200 enzymes from representative prokaryotic species, enabling inferred annotation of ∼35% of the HADSF. An extremely high level of substrate ambiguity was revealed, with the majority of HADSF enzymes using more than five substrates. Substrate profiling allowed assignment of function to previously unannotated enzymes with known structure, uncovered potential new pathways, and identified iso-functional orthologs from evolutionarily distant taxonomic groups. Intriguingly, the HADSF subfamily having the least structural elaboration of the Rossmann fold catalytic domain was the most specific, consistent with the concept that domain insertions drive the evolution of new functions and that the broad specificity observed in HADSF may be a relic of this process.

mTOR regulates peripheral nerve response to tensile strain.

While excessive tensile strain can be detrimental to nerve function, strain can be a positive regulator of neuronal outgrowth. We used an in vivo rat model of sciatic nerve strain to investigate signaling mechanisms underlying peripheral nerve response to deformation. Nerves were deformed by 11% and did not demonstrate deficits in compound action potential latency or amplitude during or after 6 h of strain. As revealed by Western blotting, application of strain resulted in significant upregulation of mammalian target of rapamycin (mTOR) and S6 signaling in nerves, increased myelin basic protein (MBP) and ß-actin levels, and increased phosphorylation of neurofilament subunit H (NF-H) compared with unstrained (sham) contralateral nerves (P < 0.05 for all comparisons, paired two-tailed t-test). Strain did not alter neuron-specific ß3-tubulin or overall nerve tubulin levels compared with unstrained controls. Systemic rapamycin treatment, thought to selectively target mTOR complex 1 (mTORC1), suppressed mTOR/S6 signaling, reduced levels of MBP and overall tubulin, and decreased NF-H phosphorylation in nerves strained for 6 h, revealing a role for mTOR in increasing MBP expression and NF-H phosphorylation, and maintaining tubulin levels. Consistent with stretch-induced increases in MBP, immunolabeling revealed increased S6 signaling in Schwann cells of stretched nerves compared with unstretched nerves. In addition, application of strain to cultured adult dorsal root ganglion neurons showed an increase in axonal protein synthesis based on a puromycin incorporation assay, suggesting that neuronal translational pathways also respond to strain. This work has important implications for understanding mechanisms underlying nerve response to strain during development and regeneration.NEW & NOTEWORTHY Peripheral nerves experience tensile strain (stretch) during development and movement. Excessive strain impairs neuronal function, but moderate strains are accommodated by nerves and can promote neuronal growth; mechanisms underlying these phenomena are not well understood. We demonstrated that levels of several structural proteins increase following physiological levels of nerve strain and that expression of a subset of these proteins is regulated by mTOR. Our work has important implications for understanding nerve development and strain-based regenerative strategies.

Control of carotenoid biosynthesis through a heme-based cis-trans isomerase.

Plants synthesize carotenoids, which are essential for plant development and survival. These metabolites also serve as essential nutrients for human health. The biosynthetic pathway for all plant carotenoids occurs in chloroplasts and other plastids and requires 15-cis-ζ-carotene isomerase (Z-ISO). It was not known whether Z-ISO catalyzes isomerization alone or in combination with other enzymes. Here we show that Z-ISO is a bona fide enzyme and integral membrane protein. Z-ISO independently catalyzes the cis-trans isomerization of the 15-15' carbon-carbon double bond in 9,15,9'-cis-ζ-carotene to produce the substrate required by the subsequent biosynthetic-pathway enzyme. We discovered that isomerization depends upon a ferrous heme b cofactor that undergoes redox-regulated ligand switching between the heme iron and alternate Z-ISO amino acid residues. Heme b-dependent isomerization of a large hydrophobic compound in a membrane was previously undescribed. As an isomerase, Z-ISO represents a new prototype for heme b proteins and potentially uses a new chemical mechanism.

Crystal structure of a phosphorylation-coupled saccharide transporter.

Saccharides have a central role in the nutrition of all living organisms. Whereas several saccharide uptake systems are shared between the different phylogenetic kingdoms, the phosphoenolpyruvate-dependent phosphotransferase system exists almost exclusively in bacteria. This multi-component system includes an integral membrane protein EIIC that transports saccharides and assists in their phosphorylation. Here we present the crystal structure of an EIIC from Bacillus cereus that transports diacetylchitobiose. The EIIC is a homodimer, with an expansive interface formed between the amino-terminal halves of the two protomers. The carboxy-terminal half of each protomer has a large binding pocket that contains a diacetylchitobiose, which is occluded from both sides of the membrane with its site of phosphorylation near the conserved His250 and Glu334 residues. The structure shows the architecture of this important class of transporters, identifies the determinants of substrate binding and phosphorylation, and provides a framework for understanding the mechanism of sugar translocation.

Crystal structure of a potassium ion transporter, TrkH.

The TrkH/TrkG/KtrB proteins mediate K(+) uptake in bacteria and probably evolved from simple K(+) channels by multiple gene duplications or fusions. Here we present the crystal structure of a TrkH from Vibrio parahaemolyticus. TrkH is a homodimer, and each protomer contains an ion permeation pathway. A selectivity filter, similar in architecture to those of K(+) channels but significantly shorter, is lined by backbone and side-chain oxygen atoms. Functional studies showed that TrkH is selective for permeation of K(+) and Rb(+) over smaller ions such as Na(+) or Li(+). Immediately intracellular to the selectivity filter are an intramembrane loop and an arginine residue, both highly conserved, which constrict the permeation pathway. Substituting the arginine with an alanine significantly increases the rate of K(+) flux. These results reveal the molecular basis of K(+) selectivity and suggest a novel gating mechanism for this large and important family of membrane transport proteins.

Systematic Assessment of Research on Autism Spectrum Disorder (ASD) and Mercury Reveals Conflicts of Interest and the Need for Transparency in Autism Research.

Historically, entities with a vested interest in a product that critics have suggested is harmful have consistently used research to back their claims that the product is safe. Prominent examples are: tobacco, lead, bisphenol A, and atrazine. Research literature indicates that about 80-90% of studies with industry affiliation found no harm from the product, while only about 10-20% of studies without industry affiliation found no harm. In parallel to other historical debates, recent studies examining a possible relationship between mercury (Hg) exposure and autism spectrum disorder (ASD) show a similar dichotomy. Studies sponsored and supported by industry or entities with an apparent conflict of interest have most often shown no evidence of harm or no "consistent" evidence of harm, while studies without such affiliations report positive evidence of a Hg/autism association. The potentially causal relationship between Hg exposure and ASD differs from other toxic products since there is a broad coalition of entities for whom a conflict of interest arises. These include influential governmental public health entities, the pharmaceutical industry, and even the coal burning industry. This review includes a systematic literature search of original studies on the potential relationship between Hg and ASD from 1999 to August 2015, finding that of the studies with public health and/or industry affiliation, 86% reported no relationship between Hg and ASD. However, among studies without public health and/or industry affiliation, only 21% find no relationship between Hg and ASD. The discrepancy in these results suggests a bias indicative of a conflict of interest.

Homologue structure of the SLAC1 anion channel for closing stomata in leaves.

The plant SLAC1 anion channel controls turgor pressure in the aperture-defining guard cells of plant stomata, thereby regulating the exchange of water vapour and photosynthetic gases in response to environmental signals such as drought or high levels of carbon dioxide. Here we determine the crystal structure of a bacterial homologue (Haemophilus influenzae) of SLAC1 at 1.20 Å resolution, and use structure-inspired mutagenesis to analyse the conductance properties of SLAC1 channels. SLAC1 is a symmetrical trimer composed from quasi-symmetrical subunits, each having ten transmembrane helices arranged from helical hairpin pairs to form a central five-helix transmembrane pore that is gated by an extremely conserved phenylalanine residue. Conformational features indicate a mechanism for control of gating by kinase activation, and electrostatic features of the pore coupled with electrophysiological characteristics indicate that selectivity among different anions is largely a function of the energetic cost of ion dehydration.

Bidirectional actin transport is influenced by microtubule and actin stability.

Local and long-distance transport of cytoskeletal proteins is vital to neuronal maintenance and growth. Though recent progress has provided insight into the movement of microtubules and neurofilaments, mechanisms underlying the movement of actin remain elusive, in large part due to rapid transitions between its filament states and its diverse cellular localization and function. In this work, we integrated live imaging of rat sensory neurons, image processing, multiple regression analysis, and mathematical modeling to perform the first quantitative, high-resolution investigation of GFP-actin identity and movement in individual axons. Our data revealed that filamentous actin densities arise along the length of the axon and move short but significant distances bidirectionally, with a net anterograde bias. We directly tested the role of actin and microtubules in this movement. We also confirmed a role for actin densities in extension of axonal filopodia, and demonstrated intermittent correlation of actin and mitochondrial movement. Our results support a novel mechanism underlying slow component axonal transport, in which the stability of both microtubule and actin cytoskeletal components influence the mobility of filamentous actin.

Experimental strategies for functional annotation and metabolism discovery: targeted screening of solute binding proteins and unbiased panning of metabolomes.

The rate at which genome sequencing data is accruing demands enhanced methods for functional annotation and metabolism discovery. Solute binding proteins (SBPs) facilitate the transport of the first reactant in a metabolic pathway, thereby constraining the regions of chemical space and the chemistries that must be considered for pathway reconstruction. We describe high-throughput protein production and differential scanning fluorimetry platforms, which enabled the screening of 158 SBPs against a 189 component library specifically tailored for this class of proteins. Like all screening efforts, this approach is limited by the practical constraints imposed by construction of the library, i.e., we can study only those metabolites that are known to exist and which can be made in sufficient quantities for experimentation. To move beyond these inherent limitations, we illustrate the promise of crystallographic- and mass spectrometric-based approaches for the unbiased use of entire metabolomes as screening libraries. Together, our approaches identified 40 new SBP ligands, generated experiment-based annotations for 2084 SBPs in 71 isofunctional clusters, and defined numerous metabolic pathways, including novel catabolic pathways for the utilization of ethanolamine as sole nitrogen source and the use of d-Ala-d-Ala as sole carbon source. These efforts begin to define an integrated strategy for realizing the full value of amassing genome sequence data.

Structural genomics for drug design against the pathogen Coxiella burnetii.

Coxiella burnetii is a highly infectious bacterium and potential agent of bioterrorism. However, it has not been studied as extensively as other biological agents, and very few of its proteins have been structurally characterized. To address this situation, we undertook a study of critical metabolic enzymes in C. burnetii that have great potential as drug targets. We used high-throughput techniques to produce novel crystal structures of 48 of these proteins. We selected one protein, C. burnetii dihydrofolate reductase (CbDHFR), for additional work to demonstrate the value of these structures for structure-based drug design. This enzyme's structure reveals a feature in the substrate binding groove that is different between CbDHFR and human dihydrofolate reductase (hDHFR). We then identified a compound by in silico screening that exploits this binding groove difference, and demonstrated that this compound inhibits CbDHFR with at least 25-fold greater potency than hDHFR. Since this binding groove feature is shared by many other prokaryotes, the compound identified could form the basis of a novel antibacterial agent effective against a broad spectrum of pathogenic bacteria.

Optimising the combination of thermostabilising mutations in the neurotensin receptor for structure determination.

Conformational thermostabilisation of G protein-coupled receptors is a successful approach for their structure determination. We have recently determined the structure of a thermostabilised neurotensin receptor NTS1 in complex with its peptide agonist and here we describe the strategy for the identification and combination of the 6 thermostabilising mutations essential for crystallisation. First, thermostability assays were performed on a panel of 340 detergent-solubilised Ala/Leu NTS1 mutants and the best 16 thermostabilising mutations were identified. These mutations were combined pair-wise in nearly all combinations (119 out of a possible 120 combinations) and each mutant was expressed and its thermostability was experimentally determined. A theoretical stability score was calculated from the sum of the stabilities measured for each double mutant and applied to develop 24 triple mutants, which in turn led to the construction of 14 quadruple mutants. Use of the thermostability data for the double mutants to predict further mutant combinations resulted in a greater percentage of the triple and quadruple mutants showing improved thermostability than if only the thermostability data for the single mutations was considered. The best quadruple mutant (NTS1-Nag36k) was further improved by including an additional 2 mutations (resulting in NTS1-GW5) that were identified from a complete Ala/Leu scan of Nag36k by testing the thermostability of the mutants in situ in whole bacteria. NTS1-GW5 had excellent stability in short chain detergents and could be readily purified as a homogenous sample that ultimately allowed crystallisation and structure determination.

Parvimonas micra Bacteremia: A Rare Complication After Esophagogastroduodenoscopy for Upper Gastrointestinal Bleeding.

Parvimonas micra bacteremia is rarely encountered in clinical practice. When it is, patients usually have underlying periodontal disease or colorectal carcinoma. To the best of our knowledge, this is the first case of P. micra bacteremia in a patient without the predisposing risk factors listed above. We postulate that this occurred because of translocation across an interrupted gut-blood barrier in the setting of an acute upper gastrointestinal bleed. We present this case to highlight the importance of identifying and treating P. micra bacteremia because it can prevent commonly encountered sequelae of untreated bacteremia and improve outcomes.

Clinical outcomes of nonvariceal upper gastrointestinal bleeding in nonagenarians and octogenarians: a comparative nationwide analysis.

BACKGROUND/AIMS: Nonagenarians will purportedly account for 10% of the United States population by 2050. However, no studies have assessed the outcomes of nonvariceal upper gastrointestinal bleeding (NVUGIB) in this age group. METHODS: The National Inpatient Sample database between 2016 and 2020 was used to compare the clinical outcomes of NVUGIB in nonagenarians and octogenarians and evaluate predictors of mortality and the use of esophagogastroduodenoscopy (EGD). RESULTS: Nonagenarians had higher in-hospital mortality than that of octogenarians (4% vs. 3%, p<0.001). EGD utilization (30% vs. 48%, p<0.001) and blood transfusion (27% vs. 40%, p<0.001) was significantly lower in nonagenarians. Multivariate logistic regression analysis revealed that nonagenarians with NVUGIB had higher odds of mortality (odds ratio [OR], 1.5; 95% confidence interval [CI], 1.3-1.7) and lower odds of EGD utilization (OR, 0.86; 95% CI, 0.83-0.89) than those of octogenarians. CONCLUSIONS: Nonagenarians admitted with NVUGIB have a higher mortality risk than that of octogenarians. EGD is used significantly in managing NVUGIB among nonagenarians; however, its utilization is comparatively lower than in octogenarians. More studies are needed to assess predictors of poor outcomes and the indications of EGD in this growing population.

Crystal structure of human Karyopherin ß2 bound to the PY-NLS of Saccharomyces cerevisiae Nab2.

Import-Karyopherin or Importin proteins bind nuclear localization signals (NLSs) to mediate the import of proteins into the cell nucleus. Karyopherin ß2 or Kapß2, also known as Transportin, is a member of this transporter family responsible for the import of numerous RNA binding proteins. Kapß2 recognizes a targeting signal termed the PY-NLS that lies within its cargos to target them through the nuclear pore complex. The recognition of PY-NLS by Kapß2 is conserved throughout eukaryotes. Kap104, the Kapß2 homolog in Saccharomyces cerevisiae, recognizes PY-NLSs in cargos Nab2, Hrp1, and Tfg2. We have determined the crystal structure of Kapß2 bound to the PY-NLS of the mRNA processing protein Nab2 at 3.05-Å resolution. A seven-residue segment of the PY-NLS of Nab2 is observed to bind Kapß2 in an extended conformation and occupies the same PY-NLS binding site observed in other Kapß2·PY-NLS structures.

Nerve strain correlates with structural changes quantified by Fourier analysis.

INTRODUCTION: Nerve deformation affects physiological function. Bands of Fontana are an optical manifestation of axonal undulations and may provide a structural indicator of nerve strain. METHODS: We developed an automated Fourier-based image processing method to quantify the periodicity of bands of Fontana both in bright field images and in axonal undulations in immunolabeled longitudinal sections. RESULTS: We found a strong linear relationship between applied strain and the frequency of bands of Fontana in rat sciatic nerves (-0.0056 µm(-) ⋅%(-) , r2 = 0.829; P < 0.05). This relationship agreed with the observed trend between strain and axonal waviness, calculated from longitudinal sections of sciatic nerves immunolabeled against myelin basic protein. CONCLUSIONS: This accurate and objective approach has potential to increase our understanding of structure-function relationships in the nervous system and to guide preservation and enhancement of neural function.